Genome-wide analysis for discovery of rice microRNAs reveals natural antisense microRNAs (nat-miRNAs)

Abstract

Small RNAs (21-24 nt) are involved in gene regulation through translation inhibition, mRNA cleavage, or directing chromatin modifications. In rice, currently approximately 240 microRNAs (miRNAs) have been annotated. We sequenced more than four million small RNAs from rice and identified another 24 miRNA genes. Among these, we found a unique class of miRNAs that derive from natural cis-antisense transcript pairs. This configuration generates miRNAs that can perfectly match their targets. We provide evidence that the miRNAs function by inducing mRNA cleavage in the middle of their complementary site. Their production requires Dicer-like 1 (DCL1) activity, which is essential for canonical miRNA biogenesis. All of the natural antisense miRNAs (nat-miRNAs) identified in this study have large introns in their precursors that appear critical for nat-miRNA evolution and for the formation of functional miRNA loci. These findings suggest that other natural cis-antisense loci with similar exon-intron arrangements could be another source of miRNA genes.

Fig. 1.

Identification and analysis of rice miRNAs and targets. (A) Flowchart for the prediction of potential miRNAs from rice MPSS data. (B) RNA gel blots of LMW RNA isolated from inflorescence tissues (I) and 2-week-old seedlings (S) probed with labeled oligonucleotides. U6 loading controls are shown in the bottom row. (C) Validation of predicted targets. The arrow indicates a site verified by 5′ RACE, with the number of cloned RACE products sequenced shown above.

Fig. 2.

Gel blots to evaluate the conservation of identified miRNAs in different plants. LMW RNAs isolated from seedlings of various plants were probed with labeled oligonucleotides.

Fig. 3.

nat-miRNA analysis. (A) Potential secondary structure of nat-miRNA precursors. The triangle indicates the ≈3-kb intron in the primary transcript. (B) Expression of nat-miRNAs. I, inflorescence tissues; S, seedlings; wt, wild-type rice seedlings; 1 and 2, seedlings from two independent DCL1 RNAi lines. The P16-aC3 siRNA is the same as described in ref. 33. (C) Alignment of nat-sm1 sequence from rice with the predicted homologs in other monocots. (D) Potential nat-sm1 precursors from other monocots. Orange triangles indicate the confirmed introns in maize and sorghum. Gray triangles indicate putative splicing sites in other precursors.

Fig. 4.

Model for nat-miRNA biosynthesis and function. The nat-miRNA pathway initiates with the splicing of pri-miRNA transcripts to yield pre-miRNA hairpins. Our data support the contention that the possession of the introns in nat-miRNA precursors is important for the biosynthesis of miRNAs. Splicing of these introns limits the potential base-pairing of the pre-nat-miRNA with the sense transcript and favors hairpin formation. After Dicer cleavage, the mature nat-miRNAs then enter the cytoplasm and direct the cleavage of the sense transcripts that are their targets. AS, antisense strand; SS, sense strand.